Ribosome inactivating proteins, which are toxic to animals and humans (RIPS, also known as ribotoxins), are generally derived from plant proteins. RIPs act by catalytically inactivating ribosomes in eukaryotic cells which inhibit protein synthesis and cause cellular destruction. RIPs are generally divided into two classes, the Type 1 RIP and the Type 2 RIP. There is significant amino acid sequence homology between members of Type 1 and Type 2 RIPs and bacterial Shiga and Shiga-like toxins, however, the Type 2 RIPs and the bacterial Shiga and Shiga-like toxins have similar mechanisms of action.
Type 2 RIPs are composed of two polypeptide chains: an A-chain, and a lectin-like protein referred to as a B-chain. The A-chain and B-chain are covalently attached to each other via a disulfide bond. The B-chain has a high affinity for cell surface moieties, and is capable of reversibly binding to a specific receptor on the cell membrane. Once bound to the specific receptor, the uptake of the A-chain into the cell is achieved through endocytosis.
Once inside the cell, the A-chain, which is an N-glycosidase, operates to enzymatically remove an adenine base at a specific site on the 28S ribosomal RNA (r-RNA). The resulting depurination action prevents an elongation factor from binding to the r-RNA, thus inhibiting protein synthesis. For these reasons, Type 2 RIPs are very potent cytotoxins and animal poisons even at extremely low concentrations. The best known Type 2 RIP is ricin which has a median lethal dose (LD50) of about 3 μg/kg.
Type 1 RIPs are typically composed of a single polypeptide chain that is equivalent in activity to the A-chain of Type 2 RIPs. Lacking analogs of the B-chain, Type 1 RIPs are minimally toxic to cells with intact membranes. However, in absence of cell membranes, Type 1 RIPs retain significant potency in inhibiting ribosomes and protein synthesis.
Type 1 and Type 2 RIPs may be derived from a variety of dicot and monocot plants, and thus can be found in many places. They are often abundant in seeds, roots and the latex of the plant. The exact function of RIPs in vivo is unclear. It has been theorized that they act as antiviral or antifungal agents. Type 2 RIPs possess the catalytically active A-chain, and retain depurination activity in ribosomes. Type 2 RIPs generally target a specific nucleotide sequence called the G(A)GA tetraloop which is typically found in the large ribosomal RNA of eukaryotic cells, and enzymatically remove the first adenine base, (A), from the tetraloop nucleotide sequence. For example, in rat liver cells, the A-chain has been shown to remove a specific adenine base through cleavage of the glycosidic bond of adenine 4323 from 28S ribosomal RNA.
Type 2 RIPs including ricin have been studied and tested for use in weapon systems. Their extreme toxicity make them potential candidates for use or deployment during warfare or acts of terrorism. In the event that Type 2 RIPs are ever deployed, part of the initial defense includes the rapid and accurate detection of RIPs especially at the submicrogram levels of concentration. Currently available military systems for detecting and identifying RIPs are laboratory based, requiring sophisticated and expensive equipment. Therefore, such systems are of limited practical use in the field.
Accordingly, there is a need to develop methods for rapidly detecting RIPs particularly Type 2 RIPs and related ribotoxins, including, but not limited to, ricin toxin A-chain (RTA), ricin, abrin, gelonin, SLT-1, momordin, which would permit protective measures or countermeasures to be quickly implemented in the event of an attack with weapons employing the same. There is also a need to provide an assay preferably in the form of a field kit that is sturdy, portable, rapidly deployable and simple to use. Moreover, the demand for methods and assays capable of rapidly detecting RIPs has applications beyond those of the military such as in the pharmaceutical, medical, food and public safety industries, and the like.